Novel cycloheptathiophene intermediates for the synthesis of biotin

ABSTRACT

A novel process for the preparation of biotin is disclosed wherein said process employs inexpensive starting materials and reagents resulting in the obtention of biotin devoid of the biologically inactive stereoisomers.

This is a division of application Ser. No. 929,870 filed July 31, 1978,now U.S. Pat. No. 4,175,086 which in turn is a divisional of Ser. No.836,876, filed Sept. 26, 1977, now U.S. Pat. No. 4,124,595, which inturn is a divisional of Ser. No. 771,218, filed Feb. 23, 1977, now U.S.Pat. No. 4,062,868.

BACKGROUND OF THE INVENTION

Biotin, vitamin H, is a natural product found largely in the kidney,liver, egg yolk, milk and yeast. The compound is used to preventsymptoms of eggwhite injury in experimental animals. Its prime medicaluse is in various dermatitides.

Biotin has been prepared synthetically by Harris et al. (Science, 97,447 (1943) and Baker et al. (J. Org. Chem. 12, 167 (1947), among others.None of these syntheses, however, were commercially feasible. The firstcommercial synthesis of biotin resulted from the work of Goldberg andSternbach (U.S. Pat. Nos. 2,489,235 and 2,489,236).

Many previous biotin syntheses suffer from the disadvantage thatstereoisomeric mixtures of intermediates leading to biotin stereoisomersare formed, thus requiring costly and time consuming separations. Theseseparations also lead to decreased yields of biotin. In the instantinvention an inexpensive and readily available starting material, i.e.,a 3-halocycloheptene, preferably 3-bromocycloheptene, is converted tobiotin in a stereospecific synthesis, resulting in the obtention ofbiotin devoid of biologically inactive stereoisomers.

According to the instant invention, biotin is obtained from a relativelyinexpensive starting material, in a stereospecific fashion thus avoidingthe costly and inefficient chemical separations heretofore required.

SUMMARY OF THE INVENTION

This invention is directed to a process for selectively synthesizingbiotin, which has the structural formula: ##STR1## from a3-halocycloheptene, a compound of the formula: ##STR2## wherein X ishalogen.

DETAILED DESCRIPTION OF THE INVENTION

As used throughout this application, the term "lower alkyl" denotesstraight chain and branched chain saturated aliphatic hydrocarbon groupshaving from 1 to 8 carbon atoms, such as methyl, ethyl and propyl,preferably methyl. As also used herein, the term "aryl" signifiesmononuclear aromatic hydrocarbyl groups of 6 to 13 carbon atoms, such asphenyl and tolyl, which can be unsubstituted or substituted in one ormore positions with a lower alkylenedioxy, a halogen, a nitro, a loweralkyl or a lower alkoxy substituent, and polynuclear aryl groups of 10to 17 carbon atoms, such as naphthyl, anthryl, phenanthryl and azulyl,which can be substituted with one or more of the aforementioned groups.The preferred aryl groups are the substituted and unsubstitutedmononuclear aryl groups, particularly phenyl. As further used herein,the term "aryl lower alkyl" comprehends groups wherein aryl and loweralkyl are as defined above, particularly benzyl. As still further usedherein, the term "lower alkoxy" comprehends groups having from 1 to 7carbon atoms such as methoxy and ethoxy. Also herein, the term "halogen"or "halo", unless otherwise stated, comprehends fluorine, chlorine,bromine and iodine. Further herein, the term "lower alkylenedioxy"comprehends lower alkylenedioxy groups having 1 to 4 carbon atoms, suchas methylenedioxy and ethylenedioxy. The term "alkali metal" as usedherein, unless otherwise stated, denotes sodium, lithium, and potassium.The term "alkaline earth metal" denotes calcium, magnesium, and barium.The term "lower acyl" as used herein, denotes acyl groups having 1-8carbon atoms (including the carbonyl carbon). As still further usedthroughout this application, in the pictorial representations of thecompounds of this application, a thickened tapered line ( ) indicates asubstituent which is in the β-orientation (above the plane of themolecule), a dotted line ( ) indicates a substituent which is in theα/orientation (below the plane of the molecule) and a wavy line ( )indicates a substituent which is in either the α- or β-orientation. Itis to be understood that the pictorial representations of the compoundsgiven throughout the specification are set forth for convenience and areto be construed as inclusive of other forms, including enantiomers andracemates, and are not to be construed as limited to the particular formshown. A black dot (•) indicates the configuration of the hydrogen atomat that stereocenter to be in the β-orientation.

In accordance with this invention, biotin is obtained by firstconverting halocycloheptene of formula I to a compound of the formula:##STR3## wherein Y is a lower acyl moiety derived from the thiolacidemployed in the preparation of compound II.

The transformation of compound I to compound II occurs by way of analkylation between compound I and a thiolacid, preferably thiolaceticacid. The aforementioned alkylation is carried out in a polar protic oraprotic solvent in the presence of a base. Typical solvents that may beemployed in this reaction are acetonitrite, benzonitrile, chlorobenzene,methylenechloride, chloroform, aniline, pyridine, acetone, loweralkanols, nitrobenzene, dimethylformamide (DMF), dimethylsulfoxide(DMSO) hexamethylphosphoric acid triamide, and the like.

In carrying out the above reaction, any conventional nitrogen containingbase may be employed. Typical of these bases are primary amines such asmethyl, or ethyl amines, aniline; secondary amines such as dimethyl,diethyl amines, pyrrole; tertiary amines such as trimethyl,triethylamines and pyridine. Inorganic bases such as alkali metalhydroxides, alkaline earth metal hydroxides and alkali metal alkoxidesmay also be employed. Although temperature and pressure are notcritical, the reaction is usually carried out at atmospheric pressureand at a temperature ranging from about -20° C. to about 50° C.,preferably at about room temperature.

Compound II is then transformed to a compound of the formula: ##STR4##wherein R is lower alkyl.

The conversion of compound II to compound III occurs by reactingcompound II with a halogenated acetal of the formula:

    X--CH.sub.2 CH(OR).sub.2

wherein X and R are as defined above; in the presence of an alkali metalalkoxide.

The transformation of compound II to compound III takes place at atemperature of from about 50° C. to the reflux temperature of thesolvent. The solvent employed may be an inert solvent such as a loweralkanol, e.g., methanol, ethanol and the like, diloweralkyl ethers suchas diethyl ether, dibutyl ether, and the like, cyclic ethers, such asdioxane and tetrahydrofuran. Solvents such as cyclohexane, benzene,toluene, xylene, pentane, heptane, hexane and the like. The alkali metalalkoxide employed is preferably sodium ethoxide.

Compound III is then hydrolyzed to a compound of the formula: ##STR5##Any conventional method of preparing an aldehyde from an acetal may beused to convert the compound of formula III to formula IV. Preferably,compound III is acid hydrolyzed in the presence of an aqueous co-solventand a strong acid. Typical aqueous co-solvents may consist of diloweralkyl ketones such as acetone, methyl ethyl ketone and the like withwater and also lower alkanols and water. Typical strong acids that maybe employed are sulfuric, phosphoric, hydrochloric, p-toluenesulfonicand methane sulfonic acids. The reaction is generally carried out atatmospheric pressure and at the reflux temperature of the solvent.

Compound IV is then transformed to a compound of the formula: ##STR6##wherein R₁ is aryl lower alkyl.

The transformation of compound IV to compound V proceeds by way of a1,3-intramolecular dipolar addition. This transformation proceeds bytreating compound IV with an aryl lower alkyl hydroxylamine, preferablybenzylhydroxylamine in the presence of a polar aprotic solvent and acatalytic amount of an amine base. The amine bases and polar aproticsolvents may be selected from those set forth hereinabove. Particularlypreferred solvents are acetonitrile and methylene chloride. Thetransformation of compound IV to compound V is particularly unique inthat there is provided a tricyclic system containing all threestereocenters of biotin in their correct relative configuration. Theconversion of compound IV to compound V is generally carried out atatmospheric pressure and at the reflux temperature of the solvent. Thisreaction is stereospecific and no other stereoisomer of V is produced.

Compound V is then converted to a compound of the formula: ##STR7##wherein R₁ is as previously defined.

The above transformation is accomplished by reduction of compound Vemploying conventional reducing agents such as Al/Hg in methanol, alkalimetal borohydrides, such as sodium and lithium borohydrides, zinc,metal, iron and stannous chlorides in the presence of strong acids.Typical strong acids such as HCl, HBr, HI, H₂ SO₄, HNO₃, H₃ PO₄,chloroacetic, trichloroacetic acid and the like. In addition, andparticularly preferred, as reducing agents are LiAlH₄,diisobutylaluminum hydride (DIBAL) and Zn metal in the presence of anadmixture of water and acetic acid. Alternatively, the foregoingreduction may be accomplished using conventional hydrogenation catalystssuch as platinum, palladium, Raney nickel and Raney cobalt. Thesecatalysts may be supported or unsupported. A particularly preferredhydrogenation catalyst is palladium on a carbon support. The reductionof compound V to VI may be carried out in either an inert organicsolvent (such as those previously mentioned herein) or theabove-mentioned aqueous co-solvents at temperatures ranging from roomtemperature to the reflux temperature of the solvent.

Compound VI is then converted to a compound of the formula: ##STR8##wherein R₂ and R₃ are lower alkyl, and R₁ is as defined above; bytreating compound VI with at least two equivalents of a hydrocarbonsubstituted haloformate. Two equivalents of the hydrocarbon substitutedhaloformates are required in order to avoid the obtention of undesirableby-products. Use of less than two equivalents results in the obtentionof a mixture of starting material VI, N-acylated and O-acylatedproducts. Typical hydrocarbon substituted haloformates that may be usedare methyl, ethyl and isopropyl chloroformates. Other hydrocarbonsubstituted chloroformates that may be used are phenyl and benzylchloroformates. The conversion is generally carried out in a loweralkanol/water/inorganic base mixture. Typical lower alkanols aremethanol, ethanol and the like. Typical inorganic bases are sodium andpotassium bicarbonate, sodium and potassium hydroxide, ammoniumhydroxide and the like. Particularly preferred is methylchloroformate inan admixture of methanol-aqueous sodium bicarbonate.

Compound VII is then converted to a compound of the formula: ##STR9##wherein R₁ is as previously defined; by treating compound VII with analkali or alkaline earth metal hydroxide in an aqueous/lower alkanolco-solvent. The reaction is generally carried out at atmosphericpressure under reflux conditions.

Compound VIII is subsequently transformed to a compound of the formula:##STR10## by treating the former compound with an alkali metal in thepresence of an organic nitrogen base. The organic nitrogen bases may beselected from those mentioned hereinbefore and ammonia. Particularlypreferred reagents for this transformation are sodium, potassium, orlithium metal in the presence of ammonia or lower alkyl primary amines.

Compound IX is then base-hydrolyzed to form a compound of the formula:##STR11## The hydrolysis of compound IX to compound X is accomplished byconventional basic hydrolysis techniques. Typical bases that may beemployed are those mentioned hereinbefore. Particularly preferred arethe alkali and alkaline earth metal hydroxides. The hydrolysis iscarried out at atmospheric pressure and under reflux conditions.

Compound X is then transformed to a compound of the formula: ##STR12##wherein Q is a conventional nitrogen protecting group. Typical of theprotecting groups that may be employed are --CO₂ R₄, --COR₄, tosyl,--CO₂-t-butyl and --COCX₃, wherein R₄ is lower alkyl and X is as definedhereinbefore.

Compound XI may be formed by reacting compound X with the correspondinglower alkyl or aryl lower alkyl monocarboxylic acid anhydrides and acidhalides from which Q is derived. Another compound that may be used toprotect the amino group is t-butyl carbonylazide. Q may also be derivedfrom lower alkyl haloformates. The reaction generally takes place in alower alkanol. A particularly preferred reaction medium ismethylchloroformate in methanol. The reaction is generally conducted atroom temperature and at atmospheric pressure. The foregoing list ofprotective groups is merely illustrative and is not intended to beexhaustive. Any conventional amino protective group may be employed toform compound XI.

Compound XI is then transformed into a compound of the formula:##STR13## wherein Q is as defined above; by treating compound XI withDMSO in the presence of a monocarboxylic acid anhydride, preferablyacetic anhydride or trifluoroacetic anhydride. It is necessary that thiscombination of reagents be employed to insure that oxidation is limitedto the hydroxy group of compound XI.

Compound XII is then transformed to a compound of the formula: ##STR14##wherein Q is as defined above.

Compound XIII can be obtained from compound XII by any conventionalmethod of preparing an oxime from a keto compound. Conventionally,compound XII may be treated with a hydroxylamine hydrohalide, preferablyhydroxylamine hydrochloride in a nitrogen containing base. The preferrednitrogen bases are amines, which may be selected from any of thosementioned hereinbefore. The reaction may be carried out at amtosphericpressure and room temperature. Further, this reaction may be carried outin an inert organic solvent. Typical inert organic solvents are any ofthose mentioned hereinbefore, particularly ether or methanol.

Compound XIII then undergoes a Beckman rearrangement to form a lactam ofthe formula: ##STR15## wherein Q is as previously defined.

Compound XIV is obtained by treating compound XIII with a strong acid.Typical strong acids that may be employed are those mentionedhereinbefore. A particularly preferred strong acid is polyphosphoricacid. This rearrangement is generally carried out at atmosphericpressure and temperatures of from about 75° C. to about 125° C.

The lactam of formula XIV may be converted to compounds of the formula:##STR16## by procedures described in Confalone et al., U.S. Pat. No.3,978,084, the disclosure of which is incorporated herein by reference.An advantage that the process of this invention offers over theaforementioned Confalone et al. procedure is that there is no need tohydrogenate a thiophene moiety in order to obtain the biotintetrahydrothiophene moiety. As can be seen from the earlier steps inthis process, the tetrahydrothiophene moiety is present at a very earlystage, thus obviating the attendant disadvantages of hydrogenation.

Compound XV may be converted to biotin by treatment with phosgene in thepresence of an aqueous base. While any conventional aqueous base may beemployed alkali metal carbonates are particularly preferred. Thisreaction can be carried out at atmospheric pressure and temperaturesvarying from about -20° C. to about +75° C., preferably about 0° C.

The lactam of the formula XIV may alternatively be prepared by compoundII undergoing a Michael addition to form a compound of the formula:##STR17##

Compound XVI is obtained by treating compound II with an alkali metalalkoxide and 1-nitro-2-acetoxyethane. This Michael addition is carriedout at atmospheric pressure and at a temperature varying from about -20°C. to the reflux temperature of solvent employed, which is usually alower alkanol, preferably absolute ethanol. This particular reactiongenerally affords a 99% yield of the Michael addition product, i.e.,compound XVI.

Compound XVI is then converted to a compound of the formula: ##STR18##by treatment of the former compound with a dehydrating agent, preferablyarylisocyanates, most preferably phenyl isocyanate, in the presence of acatalytic amount of a nitrogen base. The preferred nitrogen bases areamines which may be selected from any of the bases mentionedhereinbefore. Triethylamine is particularly preferred. This reaction iscarried out at atmospheric pressure and at a temperature of from about25° C. to about 50° C. The reaction is generally carried out in anon-polar aromatic hydrocarbon solvent, e.g., benzene, toluene, orxylene. Compound XVII is particularly unique in that it possesses in itstricyclic system two of the three biotin stereocenters. Additionally,the reaction to form compound XVII is totally stereospecific thus pavingthe way for the formation of the biologically active stereoisomer ofbiotin.

Compound XVII is then reduced to form a compound having the formula:##STR19## The compound of formula X is obtained from compound XVII bythe same reduction procedure as that employed in the reduction ofcompound V to compound VI hereinbefore.

The compound of formula X may then be transformed to the compound offormula XIV by procedures set forth hereinbefore.

The compound of formula XIII, which is the immediate precursor to thelactam of formula XIV can be prepared alternatively when compound XI isa compound of the formula: ##STR20##

Compound XIa is then transformed successively to compounds ##STR21## inaccordance with the procedures set forth hereinbefore for thepreparation of compounds XII and XIII.

Compound XIIIa may then be treated with a strong acid, preferablytrifluoroacetic acid, to form a compound of the formula: ##STR22##

Compound XVIII may then be transformed to compound XIII by placingprotecting groups onto the amino moiety of compound XVIII in the samemanner as described hereinbefore with respect to forming compound XIfrom compound X.

The advantages of employing the carbo-t-butoxy protecting groups aresurvival of the protecting group moiety while the hydroxyl moiety ofcompound X is being transformed to an oxime and insurance that theacylation is limited to the nitrogen of the amino group to the exclusionof any acylation of the hydroxyl group. In addition, this particularprotecting group is easily removable so that another nitrogen protectinggroup, which will survive the ensuing Beckmann rearrangement to compoundXIV, may be utilized on compound XIII. This last advantage is importantbecause the product yield in the Beckman rearrangement product issensitive to the protecting group.

The non-limiting examples which follow are illustrative of thisinvention. All temperatures are in degrees Centigrade.

EXAMPLE 1

To a solution of 81.5 g (0.466 mole) of 3-bromocycloheptene in 300 ml ofacetonitrile cooled to 0° was added 13.13 ml (0.466 mole) of thiolaceticacid. The system was treated dropwise with 64.55 ml (0.466 mole) oftriethylamine, during which time a precipitate of triethylaminehydrobromide separated. The cooling bath was removed and the reactionallowed to proceed at 25° for 2.0 additional hours. The reaction mixturewas partitioned between 1 N HCl/methylene chloride. The aqueous phasewas further extracted with methylene chloride. The organic extracts werepooled, dried over sodium sulfate, and evaporated. The residue wasdistilled in vacuo to afford 56.18 g (0.330 mole, 71%), b.p.64°-65°/0.25 mm of 3-acetylmercaptocycloheptene, as a colorless liquid.

EXAMPLE 2

A solution of fresh sodium ethoxide prepared from 7.6 g (0.330 gram-atommetallic sodium) in 200 ml of absolute ethanol was treated dropwise with56.18 g (0.330 mole) of 3-acetylmercaptocycloheptene in 10 ml ofabsolute ethanol. The reaction was heated under reflux for 15 minutesand then cooled to room temperature. A solution of 49.69 ml (0.330 mole)of bromoacetaldehyde diethylacetal in 30 ml of absolute ethanol wasadded dropwise. The reaction mixture was heated under reflux for 2.0hours and cooled. The precipitated sodium bromide was filtered andwashed well with absolute ethanol. The filtrate was concentrated and theresidue partitioned between ether/brine. The aqueous phase was furtherextracted with ether. The organic extracts were pooled, dried overmagnesium sulfate, and evaporated to afford 80.1 g (0.328 mole, 99%) ofpure 3-[(2,2-diethoxyethyl)thio]-1-cycloheptene, as a colorless oil.

EXAMPLE 3

A solution of 108.13 g (0.443 mole) of3-[2,2-diethoxyethyl)thio]-1-cycloheptene in 1000 ml of acetone/water,9:1 was treated with 1.1 g of p-toluenesulfonic acid hydrate and heatedunder reflux for 1.0 hour, cooled, and concentrated. The residue waspartitioned between ether/10% sodium bicarbonate. The aqueous phase wasfurther extracted with ether. The organic extracts were pooled, driedover magnesium sulfate, and evaporated to afford 75.30 g (0.443 mole,100%) of 2-[(1-cyclohepten-3-yl)thio]acetaldehyde as a colorless oil.

EXAMPLE 4

A solution of 21.3 g (0.125 mole) of2-[(1-cyclohepten-3-yl)thio]acetaldehyde in 150 ml of acetonitrile wastreated with 15.3 g (0.125 mole) of benzylhydroxylamine and 1 ml oftriethylamine. The reaction was heated under reflux for 2.0 hours,cooled, and evaporated to dryness. The residue was triturated withbenzene/ethyl acetate, 98:2, in which the product is soluble. Aninsoluble impurity was filtered off and the filtrate was concentratedand chromatographed over silica using the same solvent system forelution. The product was eluted after a less polar by-product wasobtained as 22.80 g (0.083 mole, 66%) of 2aβ,4aβ,5,6,7,8,8aβ,8bβ-octahydro-2-benzyl-2H,3H-thieno[3',4',5':3,3a,4]cyclohept[d]-isoxazoleas a pale yellow oil, which crystallized to a colorless solid. Theproduct was recrystallized from petroleum ether to give an analyticallypure sample, m.p. 56°-57° C.

EXAMPLE 5

To a suspension of 37.69 g (0.137 mole) of2aβ,4aβ,5,6,7,8,8aβ,8bβ-octahydro-2-benzyl-2H,3H-thieno[3',4',5':3,3a,4]-cyclohept[d]-isoxazolein 400 ml of acetic acid/water, 1:1 was added 37.69 g (0.577 gram-atom)of zinc. The reaction was heated at 70° for 18 hours with efficientstirring and then cooled. The zinc salts were filtered off and thefiltrate was concentrated. The residue was partitioned between 10%ammonium hydroxide/methylene chloride. The aqueous phase was furtherextracted, and the organic extracts were combimed, dried over sodiumsulfate, and evaporated to yield 36.28 g (0.130 mole, 96%) of2,3β,3aβ,5,6,7,8,8aβ-octahydro-3α-benzlamino-4H-cyclohepta[b]-thiophen4α-ol as a colorless oil.

EXAMPLE 6

A solution of 11.08 g (0.040 mole) of2,3β,3aβ,5,6,7,9,8aβ-octahydro-3α-benzylamino-4H-cyclohepta[b]thiophen4α-ol in 200 ml of methanol and 100 ml of 10% sodium bicarbonate wastreated with 6.79 ml (0.088 mole) of methyl chloroformate, After 2.0hours of stirring, an additional 3.0 ml of methylchloroformate was addedand the reaction was allowed to proceed for an additional hour. Themixture was partitioned between methylene chloride/water. The aqueousphase was further extracted with 3×200 ml of methylene chloride. Theorganic extracts were pooled, dried over sodium sulfate, and evaporatedto yield 15.65 g (0.040 mol, 100%) ofbenzyl[2,3β,3aβ,5,6,7,8,8aβ-octahydro-4α-methoxycarbonyl-4H-cyclohepta[b]thiophen-3α-yl]carbamicacid, methyl ester as a colorless oil.

EXAMPLE 7

A solution of 23.0 g (0.058 mole) of benzyl2,3,3aβ,5,6,7,8,8aβ-octahydro-4α-methoxycarbonyl-4H-cyclohepta[b]-thiophen-3α-ylcarbamic acid, methyl ester in 150 ml of methanol was treated with 75 mlof 1 N sodium hydroxide and heated under reflux for 18.0 hours. Thereaction mixture was cooled and paritioned between methylenechloride/water. The aqueous phase was further extracted. The organicextracts were pooled, dried over sodium sulfate, and evaporated to give18.0 g (0.058 mole, 100%) of3aβ,5aβ,9bβ-2H-thieno[3,4,5-de]-cyclohepta[1,3]-oxazin-2-one,decahydro-3-benzyl as a crystalline solid. The product wasrecrystallized from methanol to afford the analytical sample, m.p.153°-154°.

EXAMPLE 8

A solution of 8.0 g (26.4 mmoles) of3aβ,5aβ,9aβ,9bβ-2H-thieno[3,4,5-de]cyclohepta[1,3]oxazin-2-one,decahydro-3-benzyl in 100 ml of dry tetrahydrofuran was added to 400 mlof liquid ammonia cooled to -78° C. To this solution, 3.0 g (0.130gram-atom) of metallic sodium as added in small portions over 0.5 hour.Reaction was allowed to proceed until the blue color disappeared atwhich point 5.0 g of ammonia chloride was added. The cooling bath wasremoved and the ammonia was allowed to evaporate overnight at 25°. Theresidue was partitioned between 1 N HCl/methylene chloride. The aqueousphase was further extracted with methylene chloride. The organicextracts were pooled, dried over sodium sulfate, and evaporated todryness to yield 5.0 g (23.4 mmoles), 89%) of pure3aβ,5aβ,9bβ-2H-thieno[3,4,5-de]cyclohepta-[1,3]oxazin- 2-one, decahydroas a crystalline solid. After recrystallization from ethyl acetate, theanalytical sample melted at 197°-198°.

EXAMPLE 9

A suspension of 5.0 g (23.4 mmoles) of 3aβ, 5aβ, 9aβ,9bβ-2H-thieno[3,4,5-de]cyclohepta[1,3]oxazin-2-one, decahydro in 50 mlof 2 N sodium hydroxide was heated under reflux for 16.0 hours andcooled. The reaction mixture was partitioned between methylenechloride/1 N HCl. The aqueous phase was further extracted with methylenechloride and the organic extracts were discarded. The pH of the aqueousphase was adjusted to 10 by the addition of concentrated ammoniumhydroxide. The aqueous phase was now further extracted with 4×100 mlportions of methylene chloride. The organic extracts were pooled, driedover sodium sulfate, and evaporated to yield 2.78 g (14.9 mmoles, 64%)of 2,3β,3aβ,5,6,7,8aβ-octahydro-3α-amino-4H-cyclohepta-[b]-thieno-4α-olas a colorless oil.

EXAMPLE 10

A solution of 3.0 g (16 mmoles) of 2,3β,3αβ,5,6,7,8,8αβ-octahydro-3α-amino-4H-cyclohepta[b]thiophen-4α-ol in 80ml of methanol was treated with 32 ml of 10% sodium bicarbonate. To themilky solution was added 1.8 ml (16 mmoles) of methylchloroformate. Thereaction was allowed to proceed for 0.5 hours and then was partitionedbetween methylene chloride/water. The aqueous phase was furtherextracted. The organic extracts were pooled, dried over sodium sulfate,and evaporated to yield 3.70 g (15.1 mmoles, 94%) of2,3β,3aβ,5,6,7,8aβ-octahydro-4α-hydroxy-4H-cyclohepta[b]thiophen-3α-yl-carbamicacid, methyl ester as a crystalline solid. An analytical sample, m.p.109°-110°, was prepared by recrystallizing2,3β,3aβ,5,6,7,8aβ-octahydro-4α-hydroxy-4H-cyclohepta[b]thiophen-3α-yl-carbamicacid, methyl ester from ethyl acetate/petroleum ether.

EXAMPLE 11

Following the procedure of Example 10 but employing p-toluene sulfonylchloride instead of methylchloroformate there was obtained2,3β,3aβ,5,6,7,7aβ-octahydro-4α-hydroxy-3α-[[(4-methylphenyl)sulfonyl]-amino]-4H-cyclohepta[b]thiophene,m.p. 166°-167°. An analytical sample was prepared by recrystallizationfrom ethanol.

EXAMPLE 12

Following the procedure of Example 10 but employing acetic anhydrideinstead of methylchloroformate there was obtained2,3β,3aβ,4β,5,6,7,7aβ-octahydro-4α-hydroxy-3.alpha.-acetamido-4H-cyclohepta[b]thiophene,m.p. 146°-147°. An analytical sample was prepared by byrecrystallization from ethyl acetate.

EXAMPLE 13

Following the procedure of Example 10 but employing t-butoxycarbonylazide instead of methylchloroformate there was obtained2,3β,3aβ,5,6,7,8,8aβ-octahydro-4α-hydroxy-4H-cyclohepta[b]thiophen-3α-yl-carbamicacid, t-butyl ester, m.p. 113°-114°. An analytical sample was preparedby recrystallization from hexane.

EXAMPLE 14

A solution of 2.4 g (9.78 mmoles) of2,3β,32β,5,6,7,8,8aβ-octahydro-4α-hydroxy-4H-cyclohepta[b]thiophen3α-yl-carbamic acid, methyl ester in 45 ml of dimethylsulfoxide wastreated with 30 ml of acetic anhydride. The reaction was allowed toproceed overnight. The solvents were removed in vacuo at the pump toyield 2.4 g (9.77 mmoles, 100%) of pure2,3β,3aβ,5,6,7,8,8aβ-octahydro-4-oxo-4H-cyclohepta[b]thiophen-3α-yl-carbamicacid, methyl ester as a crystalline solid. An analytical sample, m.p.102°-103°, was prepared by recrystallizing from ethyl acetate/petroleumether.

EXAMPLE 15

Following the procedure of Example 14, the compound of Example 11 wasconverted to2,3β,3aβ,5,6,8,8aβ-octahydro-3α-[[(4-methylphenyl)-sulfonyl]amino]-4H-cyclohepta[b]thiophene-4-one,m.p. 142°-143°. An analytical sample was prepared by recrystallizationfrom ethanol.

EXAMPLE 16

Following the procedure of Example 14, the compound of Example 12 wasconverted to2,3β,3aβ,5,6,7,8,8aβ-octahydro-3α-acetamido-4-oxo-4H-cyclohepta[b]-thiophen-3α-yl-carbamicacid, t-butyl ester, m.p. 116°-117°. An analytical sample was preparedby recrystallization from ethyl acetate.

EXAMPLE 17

Following the procedure of Example 14, the compound of Example 13 wasconverted to2,3β,3aβ,5,6,7,8,8aβ-octahydro-4-oxo-4H-cyclohepta[b]thiophene, m.p.152°-153°. An analytical sample was prepared by recrystallization fromethyl acetate/petroleum ether.

EXAMPLE 18

A solution of 183 mg (0.753 mmole) of2,3β,3aβ,5,6,7,8,8aβ-octahydro-4-oxo-4H-cyclohepta[b]thiophen-3α-yl-carbamicacid, methyl ester in 5 ml of ethanol and 0.2 ml of pyridine was treatedwith 69 mg (1.0 mmole) of hydroxylamine hydrochloride. The reaction washeated under reflux for 0.5 hours and cooled. The mixture waspartitioned between methylene chloride/1 N HCl. The aqueous phase wasfurther extracted. The organic extracts were combined, dried over sodiumsulfate, and evaporated to yield 173 mg (0.670 mmole, 89%) of pure2,3β,3aβ,5,6,7,8aβ-octahydro-4-hydroxyimino-4H-cyclohepta[b]thiophen-3α-yl-carbamicacid methyl ester as a crystalline solid. An analytical sample, m.p.123°-124° was prepared by recrystallizing from ethyl acetate/pentane.

EXAMPLE 19

Following the procedure of Example 18, the compound of Example 15 wasconverted to2,3β,3aβ,5,6,7,8,8aβ-octahydro-3α-[[(4-methylphenyl)sulfonyl]amino]-4H-cyclohepta[b]thiophen-4-one,antioxime, m.p. 181°-182°. An analytical sample was recrystallized fromethanol.

EXAMPLE 20

Following the procedure of Example 18, the compound of Example 16 wasconverted to2,3β,3aβ,3aβ,5,6,7,8,8aβ-octahydro-3α-acetamido-4-oxo-4H-cyclohepta[b]thiophene,antioxime, m.p. 213°-214° (ethyl acetate). An analytical sample wasrecrystallized from ethyl acetate.

EXAMPLE 21

Following the procedure of Example 18, the compound of Example 17 wasconverted to2,3β,3aβ,5,6,7,8,8aβ-octahydro-4-hydroxy-imino-4H-cyclohepta[b]thiophen-3α-yl-carbamicacid, t-butyl ester, m.p. 167°-168° (ethyl acetate/hexane). Ananalytical sample was recrystallized from ethyl acetate/hexane.

EXAMPLE 22

A mixture of 350 mg (1.35 mmoles) of2,3β,3aβ,5,6,7,8,8aβ-octahydro-4-hydroxyimino-4H-cyclohepta[b]thiophen-3α-yl-carbamicacid, methyl ester in 10.0 g of polyphosphoric acid was mechanicallystirred at 100° for 15.0 minutes. The reaction mixture was hydrolyzed byice water and then partitioned between brine/methylenechloride:methanol, 9:1. The aqueous phase was further extracted withmethylene chloride/methanol, 9:1. The organic extracts wre pooled, driedover sodium sulfate, and evaporated to leave a 160 mg residue. Thismaterial was chromatographed on 2 thick layer silica plates using ethylacetate as the eluent. The product,3-amino-4[N-carbomethoxyamino]tetrahydro-2-thiophenevaleric acid lactamwas isolated at R_(f) =0.2 and was obtained as 69 mg (0.27 mmole, 20%)of a crystalline solid which recrystallized from ethyl acetate andmelted at 242°-243°.

EXAMPLE 23

A solution of 50 mg. (0.14 mmole) of2,3β,3aβ,5,6,7,8,8aβ-octahydro-3α-[[(4-methylphenyl)sulfonyl]amino]-4H-cyclohepta[b]thiophen-4-one,antioxime in 3.7 g of polyphosphoric acid (PPA) was mechanically stirredat 50° for 8.0 hours. The PPA was hydrolyzed and the product mixture wasextracted with methylene chloride/methanol, 4:1. The organic extractswere combined, dried over sodium sulfate, and evaporated to yield 30 mgof residue. The material was chromatographed on thick layer silicaplates using ethyl acetate as the eluent. The product was isolated atR_(f) =0.4 and amounted to 15 mg (30%) of pure all cis-4-p-toluenesulfonamido-3-aminotetrahydrothiophen-2-valeric acid lactam, m.p. 210°(dec).

EXAMPLE 24

A suspension of 90 mg (0.349 mmole) of 3-amino-4-N-carbomethoxyaminotetrahydro-2-thiophenevaleric acid lactam in 15 ml of water was treatedunder argon with 3.0 g of barium hydroxide monohydrate. The reactionmixture was heated under reflux for 20 hours, cooled, and the bariumsalts were filtered off and washed with water. The filtrate wasconcentrated, cooled to 0° and treated with gaseous phosgene until thesolution was acidic to congo red. The reaction was allowed to stand for1.5 hours at 25° and then was evaporated to dryness. This residue, whichis a mixture of biotin and inorganic salts, is taken up in 15 ml of drymethanol and treated with two drops of concentrated sulfuric acid. Themixture is heated under reflux for 1.5 hours and then cooled to 25°. Theinorganic salts are filtered off and washed with chloroform/methanol,4:1. The filtrate was partitioned between water/chloroform: methanol,4:1. The aqueous phase was further extracted with chloroform/methanol,4:1. The organic extracts were combined, dried over sodium sulfate andevaporated to yield 45 mg (0.174 mmole, 50%) of pure biotin methylester, m.p. 132°-133°, after recrystallization from ethyl acetate. Theconversion of biotin methyl ester to biotin was accomplished accordingto the procedure of V. du Vigneaud¹.

EXAMPLE 25

To 40 ml of absolute ethanol was added 1.38 g (0.060 gram atom) metallicsodium. When the reaction was completed, 10.2 g (0.060 mole) of3-acetylmercaptocycloheptene in 20 ml of absolute ethanol was added andthe reaction mixture was brought up to reflux for 15 minutes. Thesolution was then cooled to 0° and 7.98 g (0.060 mole) of1-nitro-2-acetoxyethane in 20 ml absolute ethanol was added. Thereaction was allowed to proceed for 3.0 hours at 0° and then waspartitioned between 1 N HCl and methylene chloride. The aqueous phasewas further extracted with methylene chloride. The organic phases werepooled, dried over sodium sulfate, and evaporated to afford 12 g (99%)of pure 3-[2-nitroethylthio]cycloheptene as a colorless oil.

EXAMPLE 26

A solution of 15.2 g (0.075 mole) of 3-[2-nitroethylthio]-cycloheptenein 200 ml of dry benzene was treated with 24 ml (0.224 mole) ofphenylisocyanate and 0.5 ml of triethylamine. The mixture was stirred 24hours at room temperature. The white solid (carbanilide) which hadseparated was filtered off and washed well with benzene. The filtratewas evaporated to dryness and residue was chromatographed over one kg.silica eluting with benzene/ethyl acetate (98:2). Fractions containingthe product were combined and evaporated to afford 10.8 g (0.060 mole,79%) of pure3,4aβ,5,6,7,8,9β,9aβ-octahydrocycloheptano[5,5a,6-f,g]-thieno[3,4-c]isoxazoleas a colorless oil.

EXAMPLE 27

A solution of 3.66 g (0.020 mole) of3,4aβ,5,6,7,8,9β,9aβ-octahydrocycloheptane[5,5a,6-f,g]thieno[3,4-c]isoxazolein 150 ml of anhydrous ether was added dropwise at 25° (water bath)under argon to 1.64 g (0.043 mole) lithium aluminum hydride suspended in50 ml of anhydrous ether. The mixture was refluxed for 4.0 hours,cooled, and quenched dropwise with 50 ml. concentrated sodium sulfate.The reaction was extracted four times with ether. The organic extractswere combined, dried over sodium sulfate, and evaporated to yield 3.43 g(0.018 mole, 92%) of2,3β,3aβ,5,6,7,8,8aβ-octahydro-3α-amino-4H-cyclohepta[b]thiophen-4α-olas a colorless oil. For characterization, the compound was converted toits hydrochloride salt by methanolic hydrogen chloride to afford a puresample of2,3β,3aβ,5,6,7,8,8aβ-octahydro-3α-amino-4H-cyclohepta[b]thiophen-4α-ol,hydrochloride, m.p. 191°-193°. Recrystallization from ethanol/etheryielded an analytical sample, m.p. 192°-193°.

EXAMPLE 28

A solution of 3.0 g (0.010 mole) of2,3β,3aβ,5,6,7,8,8aβ-octahydro-4-hydroxyimino-4H-cyclohepta[b]thiophen-3α-yl-carbamicacid, t-butyl ester in 10 ml of trifluoroacetic acid was stirred at 0°for 1.5 hours. The solvent was evaporated and the residue wasrecrystallized from methanol-ether to yield 2.3 g (0.0073 mole, 73%) ofpure2,3β,3aβ,5,6,7,8,8aβ-octahydro-3α-amino-4-(4H)-cyclohepta[b]thiophene,antioxime trifluoracetate, m.p. 180°-181°, as a fluffy white solid.

EXAMPLE 29

A solution of 0.628 g (0.002 mole) of2,3β,3aβ,5,6,7,8,8aβ-octahydro-3α-amino-4-(4H)-cyclohepta[b]thiophene,antioxime trifluoroacetate in 4 ml. pyridine and 0.3 ml. trifluoroaceticacid anhydride was stirred at 25° for 15 minutes and then partitionedbetween 1 N HCl/methylene chloride. The aqueous phase was furtherextracted with methylene chloride. The organic phases were combined,dried over sodium sulfate, and evaporated to yield 0.590 g (0.002 mole,100%) of pure2,3β,3aβ,5,6,7,8,8aβ,-octahydro-3α-trifluoroacetamido-4-oxo-4H-cyclohepta[b]thiophene,antioximie, m.p. 213°-214°. The compound may be recrystallized fromethyl acetate.

We claim:
 1. A compound of the formula: ##STR23## wherein Q is selectedfrom the group consisting of hydrogen, --COR₄, --COCX₃ and tosyl whereR₄ is lower alkyl and X is halogen, the racemates and optical antipodesthereof.
 2. The compound of claim 1 wherein Q is ##STR24##
 3. A compoundof the formula: ##STR25## the acid salts thereof, the racemates andoptical antipodes thereof.